Tomasselli A G, Noda L H
Eur J Biochem. 1979 Jan 15;93(2):263-7. doi: 10.1111/j.1432-1033.1979.tb12819.x.
Kinetic and equilibrium dialysis substrate binding studies have been done to investigate the properties of mitochondrial GTP-AMP phosphotransferase. The results show that the enzyme has a specific requirement for divalent metal ions, namely Mg2+, Mn2+ or Ca2+ (Ca2+ is active only in the forward direction, the direction of formation of ADP). The reaction rate depends upon the ratio [Mg2+]:[substrate] rather than on the metal ion concentration alone. The enzymatic activity is influenced by NaCl (or KCl) and optimum pH occurs at 11.5 and 9.5 for guanosine and inosine nucleotides respectively. Examination of binding of substrates to the enzyme showed that there is one binding site (GTP site) for MgGTP, GTP, MgGDP or GDP per molecule of enzyme, with dissociation constants of 4.5, 4.4, 3.0, 2.2 micron respectively and one binding site (AMP site) for AMP, ADP or ATP per molecule of enzyme with dissociation constants of 20.9, 33.4 and 33.4 microns respectively. Since, within the limitations of equilibrium dialysis used in the present studies, AMP binding to one site of the enzyme could be detected only when GDP or GTP is present, the mechanism of the forward reaction may be assumed to be nearly ordered. For the reverse reaction there is no requirement of order of binding of the two nucleotides and so the mechanism of reaction may be assumed to be random.
已进行动力学和平衡透析底物结合研究，以探究线粒体GTP-AMP磷酸转移酶的性质。结果表明，该酶对二价金属离子有特定需求，即Mg2+、Mn2+或Ca2+（Ca2+仅在正向反应中具有活性，即ADP形成的方向）。反应速率取决于[Mg2+]与[底物]的比例，而非仅取决于金属离子浓度。酶活性受NaCl（或KCl）影响，鸟苷和肌苷核苷酸的最佳pH分别为11.5和9.5。对底物与酶结合的检测表明，每分子酶对MgGTP、GTP、MgGDP或GDP有一个结合位点（GTP位点），解离常数分别为4.5、4.4、3.0、2.2微米，每分子酶对AMP、ADP或ATP有一个结合位点（AMP位点），解离常数分别为20.9、33.4和33.4微米。由于在本研究中使用的平衡透析的限制范围内，只有当存在GDP或GTP时才能检测到AMP与酶的一个位点结合，因此可以假定正向反应的机制近乎有序。对于逆向反应，两种核苷酸的结合顺序没有要求，因此可以假定反应机制是随机的。
